Biology subjects

Tinti, M.

Publications and source records attributed to Tinti, M..

14 recordsLinked to original sources

bioRxiv2026

ZNK1 senses zinc and degrades zinc transporter mRNA in trypanosomes

Like other cells, parasitic and other trypanosomatids sense and regulate Zn2+ transport, but the mechanisms involved remained unknown. Here we identify a trypanosome RNA-binding protein which specifically eliminates ZIP3 Zn2+-transporter mRNA in Zn2+-replete conditions. We first demonstrated that Trypanosoma brucei ZIP3 mRNA abundance is subject to 3-untranslated region (3-UTR) and Zn2+-dependent negative control. A genome-wide RNA interference library screen, using a reporter associated with the ZIP3 3-UTR, identified Tb927.11.9510 as a candidate Zn2+-sensor. We name this protein Zinc Nuclear Knuckles 1 (ZNK1) since it localises to the nucleus and contains several Zn2+-knuckle motifs. ZNK1 is conserved among trypanosomatids, and a PIN-domain suggests a ribonuclease-based mechanism. We validate ZNK1 as a ZIP3 3-UTR dependent negative regulator and identify a GU-repeat motif in the ZIP3 3-UTR that is predictive of negative control by ZNK1. We use Cas9-editing to knockout ZNK1, and RNA-seq to assess the consequences, revealing highly specific accumulation of ZIP3 transcripts in znk1-null cells. We conclude that ZNK1 senses Zn2+-abundance and eliminates ZIP3 mRNA in a Zn2+-dependent manner. We suggest that trypanosomatid ZNK1 is an RNA-specific zinc finger nuclease that binds ZIP3 3-UTRs and degrades ZIP3 mRNA only when the tandem sensor modules are coordinated with Zn2+. Key pointsO_LITrypanosome Zinc Nuclear Knuckles 1 (ZNK1) is a zinc-sensor that eliminates zinc transporter mRNA. C_LIO_LIZNK1 negative control operates via the transporter mRNA 3-untranslated region. C_LIO_LIThe findings indicate that trypanosomatid ZNK1 is a conserved RNA-specific zinc finger nuclease. C_LI

microbiology↗

bioRxiv2026

Thermo-sensing and argonaute-dependent transcriptome remodelling in trypanosomes

Vector-borne parasites, such as African trypanosomes, experience temperature fluctuation due to fever symptomatic of infection, during developmental life cycle transitions, or due to diurnal shift. Mechanisms underpinning RNA-based thermo-sensing remain largely uncharacterised in these and other eukaryotic cells, however. Notably, trypanosomes exhibit almost exclusive polycistronic transcription, such that gene expression controls are dominated by post-transcriptional mechanisms mediated by mRNA-binding proteins and thousands of mRNA 3-untranslated regions (3-UTRs). Here, we quantify the transcriptomes and proteomes of bloodstream form Trypanosoma brucei following growth at 34{degrees}C, 37{degrees}C or 40{degrees}C for six hours. Approximately fifty genes encoding classical (co-)chaperones, with (UUA)n-rich 3-UTRs, display the expected heat-shock response. The expression of approximately 1,000 additional transcripts is also correlated with temperature, and these transcripts have relatively long 3-UTRs enriched in potentially complementary poly-purine tracts, poly-pyrimidine tracts, and palindromic sequences (P5-UTRs). To assess the potential impacts of mRNA secondary structure transcriptome-wide, we quantify mRNAs in cells lacking the central RNA interference nuclease argonaute (AGO1). Strains lacking AGO1 display increased retroposon expression, as expected, and strikingly abrogated thermo-regulation of transcripts with P5-UTRs. Thus, thermo-sensing involves argonaute-dependent transcriptome-remodelling in trypanosomes. We propose a post-transcriptional zipper hypothesis whereby access to regulatory motifs is controlled by temperature-sensitive mRNA secondary structure.

microbiology↗

bioRxiv2025

Origins and consequences of kinetoplast loss in trypanosomes

The kinetoplast is the large mitochondrial genome present in the eponymous Kinetoplastida. Trypanosoma brucei is an African trypanosome that can lose kinetoplast DNA (kDNA), however, when the nuclear-encoded gamma subunit of the mitochondrial F1FO-ATP synthase ({gamma}ATPase) is mutated. These mutations, analogous to a broken camshaft at the core of the ATP synthase rotary motor, are associated with multidrug resistance, and correlated with tsetse-fly independent mechanical transmission, and geographical spread of these parasites beyond Africa. Here we engineer kinetoplast-independent T. brucei to explore origins and consequences of kDNA loss. We used oligo targeting to edit the native{gamma} ATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identified novel M282F, M282W, and M282Y mutants, and subsequently generated precision-edited strains expressing the previously described L262P or A273P mutants, or the novel M282F mutant. Heterozygous M282F mutants retained sensitivity to the kDNA-targeting drug acriflavine, while homozygous M282F mutants were acriflavine resistant and readily tolerated acriflavine-induced kDNA loss. Proteomics analysis of the homozygous mutant pre-kDNA-loss revealed highly specific depletion of ATP synthase-associated proteins, but not the F1 subunits. Complete kDNA-loss in these cells was associated with substantial depletion of kDNA-binding proteins and mitochondrial RNA-processing factors. In contrast, mitochondrial membrane-associated transporters were increased in abundance. We conclude that T. brucei cells with a homozygous{gamma} ATPase M282F mutation assemble a remodelled ATP synthase and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome Author summaryMutations in the gamma subunit of the mitochondrial ATP synthase in parasitic African trypanosomes can have major consequences. Specifically, the entire large and complex mitochondrial genome, the kinetoplast, is rendered dispensable, and the cells become resistant to important kinetoplast-targeting drugs. Veterinary parasites with these mutations have also spread outside Africa through simple mechanical transmission, either sexually or by biting flies or vampire bats. We precision-edited the gamma subunit to replicate previously described mutants and identified a novel mutant that readily tolerated kinetoplast loss. Using quantitative proteomics, we demonstrated highly specific depletion of ATP synthase-associated proteins pre-kinetoplast-loss. We then use genome sequencing to show that the kinetoplast could be completely lost by these cells and demonstrated that cells lacking mitochondrial nucleic acids displayed specific depletion of mitochondrial nucleic acid-binding proteins. Notably, several mitochondrial membrane-associated transporter complexes were increased in abundance. Thus, we establish a method to test precise {gamma}ATPase mutations and to identify new mutations associated with kinetoplast loss. We also show that trypanosomes with a dispensable kinetoplast specifically remodel the ATP synthase pre-kinetoplast-loss and substantially remodel the mitochondrial proteome post-kinetoplast-loss.

microbiology↗

bioRxiv2025

Acoziborole resistance associated mutations in trypanosome CPSF3

Acoziborole is a safe, single dose, oral therapy, for treatment of both early and late-stage sleeping sickness, a deadly disease caused by African trypanosomes. Other benzoxaboroles show efficacy against other trypanosomatids, apicomplexans, fungi, bacteria, and viruses. Acoziborole targets the trypanosome pre-mRNA processing endonuclease, cleavage and polyadenylation specificity factor 3 (CPSF3), and triggers CPSF3 degradation, but it remains unclear whether additional mechanisms contribute to efficacy. We used oligo targeting for site saturation mutagenesis of the native CPSF3 gene. Among >1,500 edits around the putative drug binding site, only Asn232His edits conferred moderate resistance to acoziborole. Using a novel combinatorial oligo targeting method we edited multiple sites simultaneously, including sites that differ in human CPSF3, and found that an Asn232His, Tyr383Phe, Asn448Gln triple-mutant strain was >40-fold resistant to acoziborole. We used gene tagging to show that all three edits were on the same allele, and to show that triple-mutant CPSF3 was highly resistant to rapid acoziborole and proteasome-dependent degradation. Computational modelling revealed how the combinatorial mutations can disrupt acoziborole - CPSF3 interactions by introducing steric clash and by disrupting hydrophobic and water-mediated interactions. We conclude that acoziborole safety and efficacy can be explained by selective affinity for, and rapid turnover of, trypanosome CPSF3. Author SummaryDiagnosis and treatment options, previously limited for sleeping sickness, have been transformed in recent years. Acoziborole, for example, is a new, safe, single dose, oral therapy for the treatment of this deadly disease. This drug can also be used without the need for cumbersome disease-stage diagnosis. Additional boron-based drugs also show great promise against a whole range of other infectious diseases. Acoziborole targets an RNA processing enzyme in African trypanosomes, and triggers its degradation, but human cells express a similar enzyme, and alternative trypanosomal targets have also been suggested. Insights into how a drug interacts with its target can help to understand selective action against a pathogen, and to predict resistance, an ever-present threat for many drugs. We used a precision gene editing method to change the target protein in trypanosomes, editing single sites or multiple sites simultaneously. A novel triple-mutant was found to be both highly resistant to acoziborole and highly resistant to rapid degradation. Using computational models, we were able to explain how multiple mutations interfered with acoziborole binding to its target. The findings show how selective binding of a specific parasite enzyme makes acoziborole such a safe and effective drug.

microbiology↗

bioRxiv2025

Decoding efficacy and resistance space at a drug binding site

Interactions between drugs and their targets impact efficacy and, when altered by mutation, can result in resistance 1-3. Assessing and understanding the impacts of all possible mutations at a drug binding site remain challenging, however 4-6. Here, we used Multiplex Oligo Targeting (MOT) for mutational profiling, and computational modelling, to decode efficacy and resistance space at the otherwise native binding site for a low nanomolar potency, anti-trypanosomal, proteasome inhibitor 7. We saturation-edited twenty codons in the Trypanosoma brucei proteasome {beta}5 subunit and subjected the resulting MOT libraries to stepwise drug selection. Amplicon sequencing, and codon variant scoring, yielding dose-response profiles for >100 resistance-conferring mutants, among 1,280 possible codon variants. Codon variant scores were predictive of relative resistance observed using a bespoke set of mutants, while fitness profiling revealed otherwise extensive constraints on mutational fitness and resistance space. The resistance profile that emerged allowed us to readily predict routes to spontaneous drug resistance observed within accessible, single nucleotide mutational space. In silico analysis of {beta}5 subunit mutations predicted impacts on ligand affinity via steric effects, hydrogen-bonding and lipophilicity, which when combined with predictions of proteasome function perturbing mutations, were closely aligned with observed impacts on drug resistance. We conclude that MOT-library profiling facilitates assessment of all possible mutations at a drug binding site. Further decoding of drug target structure-activity relationships and drug resistance space will facilitate the design of more effective and durable drugs.

molecular biology↗

bioRxiv2025

Tissue atlas of Cryptosporidium parvum infection reveals contrasts between the natural neonatal calf model and laboratory mouse models

Cryptosporidium is an apicomplexan parasite that causes diarrhoeal disease. The species C. parvum is zoonotic and causes significant morbidity and mortality for both humans and farm animals; most commonly, calves and lambs. A One Health approach that integrates human, animal and environmental health perspectives is required to tackle this disease. Current treatments are limited and ineffective, meaning there is an urgent need to develop new anti-cryptosporidials both for human and animal health. The neonatal calf model is a natural model of infection employed as a tool for drug discovery or generating parasite material. However, the model is seldom utilised to investigate host-parasite interaction. Fundamental information about this model, including the location of the parasite in the gut, is lacking. It is also unclear how the more commonly utilised immunocompromised mouse models of cryptosporidiosis compare to the neonatal calf model. To address this, we established an acute, moderate experimental C. parvum infection in neonatal calves. Using transgenic parasites, we created a tissue atlas of infection for neonatal calf gut and immunocompromised mouse models and mapped and quantified infection to draw robust comparisons between models. Cryptosporidium infection was observed at high levels throughout the neonatal calf gastrointestinal tract and was not limited to the ileal-cecal junction, as previously suggested. This infection pattern is most similar to the acute cryptosporidiosis mouse model, interferon-gamma knockout mice (IFN{gamma}KO). Infection with transgenic parasites allowed us to perform in vivo and ex vivo tissue imaging of the chronic cryptosporidiosis mouse model, NOD SCID Gamma KO (NSG) mice. In contrast, in NSG mice infection is low in the small intestines and highest in the caecum and colon. Understanding the true distribution of infection in the gastrointestinal tract of these three key animal models provides new perspectives on how to interpret and design drug efficacy studies and provides new insight into host-pathogen interaction.

microbiology↗

bioRxiv2025

A non-coding role for trypanosome VSG transcripts in allelic exclusion

Bloodstream-form African trypanosomes display mono-telomeric expression of a Variant Surface Glycoprotein (VSG) gene in an inter-chromosomally bridged transcription and splicing compartment, such that the dominant gene produces 10,000 times more transcript than excluded VSG genes. Antigenic variation, whereby parasites switch to express other VSGs, then underpins a robust host immune evasion strategy. Specific chromatin and RNA-associated factors are required to maintain VSG exclusion, but our understanding of the mechanisms involved remains incomplete. Here we show that the VSG transcript impacts allelic competition. We induced either specific translation blockade by recruiting MS2 coat protein to the active VSG 5-untranslated region, or VSG transcript depletion using RNA interference. Neither perturbation substantially compromised exclusion of native VSGs, as determined by transcriptomic analyses. In contrast, exclusion of a VSG transgene was compromised when the native transcript was transiently depleted. Notably, while both perturbations blocked cytokinesis, an additional round of DNA replication and mitosis was observed when the transcript, known to be stabilized by a bloodstream-form specific cyclin-like F-box protein, was translationally blocked. We conclude that the VSG transcript is a bi-functional coding and non-coding RNA that participates in allelic competition to establish exclusion. Significance statementAllelic exclusion mechanisms underpin immune evasion in parasites and olfaction in mammals but the mechanisms responsible remain mysterious. VSG exclusion factors have been identified in trypanosomes, while RNA has been implicated in olfactory receptor exclusion, and in var gene exclusion in the parasites that cause malaria. The current study demonstrates a role for RNA in VSG exclusion in trypanosomes.

molecular biology↗

bioRxiv2025

Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes

Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. RNA polymerase II promoters are unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their highly divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing novel mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 native copies of histone H4, complementing the tandem arrays with a single ectopic H4 gene. The resulting histoneH4 strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine (K) residues and used multiplex amplicon-seq to profile the relative fitness of 384 distinct precision edited mutants. H4K10 mutations were not tolerated, but we could derive a panel of nineteen strains exclusively expressing novel H4K4 or H4K14 mutants. Both proteomic and transcriptomic analysis of H4K4Q mutants revealed significantly reduced expression of genes adjacent to RNA polymerase II promoters, where the glutamine (Q) mutation mimics an abnormally high level of acetylation. Thus, we present direct evidence for polycistronic expression control by histone H4 N-terminal tails in trypanosomes.

molecular biology↗

bioRxiv2024

A high-throughput protein tagging toolkit that retains endogenous UTRs for studying gene regulation in Kinetoplastids.

Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in Kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six Trypanosoma brucei proteins and demonstrate: (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.

molecular biology↗

bioRxiv2024

Cryptosporidium Oocyst Wall Proteins are true oocyst wall proteins, with COWP8 functioning to hold the inner and outer layers of the oocyst wall together

Cryptosporidiosis is a significant cause of diarrhoeal disease contributing to substantial morbidity and mortality for the immunocompromised and for young children, especially those who are malnourished. There are no vaccines available and no effective treatments for these patients. Another challenge is that Cryptosporidia are waterborne and resistant to common water treatments including chlorination. Cryptosporidia are transmitted as an oocyst that is made up of a hardy oocyst wall that protects four parasites. Little is understood about how the oocyst is constructed, its composition, and the how it resists chlorination. A family of predicted Cryptosporidium Oocyst Wall Proteins (COWPs) was identified from the genome. Using a genetic approach, we confirm that all members of the COWP family localise to the oocyst wall. Our studies indicate that COWP2, 3 and 4 localise specifically to the oocyst "suture", a zipper-like structure on the oocyst wall from which parasites emerge during infection. In parasites lacking COWP8, we observe that the inner and outer layers of the oocyst wall are no longer associated suggesting a role for COWP8 in oocyst wall morphology. Despite loss of COWP8, these transgenic parasites are viable, unchanged in mechanical strength, and retain resistance to chlorination. This work sets the foundation for future exploration of Cryptosporidium transmission.

microbiology↗

bioRxiv2023

Association of mitochondrial fucosyltransferase TbFUT1 with the assembly of the mitochondrial FoF1-ATP synthase in bloodstream form Trypanosoma brucei.

The gene TbFUT1 encodes an essential fucosyltransferase which, unexpectedly, localises to the mitochondrion of the protist parasite Trypanosoma brucei. The expression of TbFUT1 is required for the maintenance of mitochondrial membrane potential ({Psi}{Delta}m) in the bloodstream form (BSF) of the parasite, but the precise functions of TbFUT1 are unknown. Here, we demonstrate that depletion of TbFUT1 causes the accumulation of dyskinetoplastid cells; i.e., cells lacking concatenated complexes of mini- and maxicircle kinetoplast DNA (kDNA), the mitochondrial DNA of these organisms. Morphological analysis by serial face block-scanning electron microscopy showed that the dyskinetoplastid mitochondria were otherwise unperturbed with respect to structure and volume. Proteomics analyses showed that TbFUT1 depletion caused a decrease in the steady-state levels of several subunits of the Fo-subcomplex and peripheral stalk components of the mitochondrial FoF1-ATP synthase, as well as a pronounced reduction in mitochondrial ribosomal large subunit (LSU) proteins and more minor reduction in small subunit (SSU) proteins. TbFUT1 was rendered redundant with respect to cell survival and {Psi}{Delta}m generation upon F1-{gamma}WT/L262P mutation; a mutation that allows the generation of {Psi}{Delta}m in the absence of mitochondrial translation. Additionally, depletion of TbFUT1 no longer perturbs kDNA replication in these cells, indicating that dyskinetoplasty is a downstream consequence of impaired {Psi}{Delta}m. Depletion of TbFUT1 in wild type cells leads to the collapse of {Psi}{Delta}m via a functional FoF1-ATP synthase complex. We therefore conclude these mutants are inhibited in the synthesis of Fo-subcomplex components and, thus, impairing the assembly of functional FoF1-ATP synthase complexes. Curiously, mitochondrial transcript levels exhibit similar changes in abundance after FUT1 ablation in the parental and F1-{gamma}WT/L262P mutants. Further, the [~]5-fold overexpression of TbFUT1 in the TbFUT1 conditional knockout mutant under permissive conditions selectively inhibits the formation of the fully RNA-edited A6 transcript by an unknown mechanism, partially suppressing FoF1-ATP synthase assembly in these mutants. Together, these data suggest that mitochondrial fucosylation is essential for the assembly of protein complexes containing kDNA encoded subunits.

molecular biology↗

bioRxiv2021

Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation. Author SummaryAfrican trypanosomes are single-celled extracellular parasites of humans and animals relying on essential host nutrients for survival. They satisfy their iron needs by capturing host transferrin-bound iron using a surface-localised transferrin receptor (TfR) that is structurally distinct from its host counterpart. Little is known about the trypanosome response to fluctuations in host iron availability, with the exception of modulated TfR expression. We show that unlike other eukaryotes, at the transcriptome level, trypanosomes do not regulate iron-dependent enzymes as a mechanism to cope with iron deprivation. Instead, we identify a group of novel iron responsive trypanosome-specific genes, particularly an RNA Binding Protein RBP5 that is responsive to iron levels, albeit mediated by a distinct mechanism from TfR. We show that although RBP5 expression is elevated at the mRNA and protein levels, increased abundance above a maximum threshold is toxic. The trypanosome TfR has been suggested as a therapeutic target, but whether it is essential for optimal host colonisation is unclear. Our data demonstrate that trypanosomes efficiently import free iron from their environment independent of TfR suggesting that alternative iron uptake pathways exist, and that any therapeutic interventions targeting TfR must be evaluated with caution.

microbiology↗

bioRxiv2020

Proteomic identification of the UDP-GlcNAc : PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of .

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc : PI 1-6 GlcNAc-transferase. This enzyme has been shown to be a complex of at least seven subunits in mammalian cells and a similar complex of homologous subunits has been postulated in yeast. Homologs of most of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and partner proteins TbGPI15, TbGPI9, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc : PI 1-6 GlcNAc-transferase complex. Graphical abstractFirst step of GPI anchor biosynthesis pathway in T.brucei BSF is catalysed by TbGPI3 complex. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

pathology↗

bioRxiv2020

Genome-scale functional profiling of cell cycle controls in African trypanosomes

Trypanosomatids, which include major pathogens of humans and livestock, are divergent eukaryotes for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle regulators in bloodstream form Trypanosoma brucei. We induced massive parallel knockdown and sorted the perturbed population into cell cycle stages using flow cytometry. RNAi-targets were deep-sequenced from each stage and cell cycle profiles were digitally reconstructed at a genomic scale. We identify hundreds of proteins that impact cell cycle progression; glycolytic enzymes required for G1S progression, DNA replication factors, mitosis regulators, proteasome and kinetochore complex components required for G2M progression, flagellar and cytoskeletal components required for cytokinesis, mRNA-binding factors, protein kinases and many previously uncharacterised proteins. The outputs facilitate functional annotation and drug-target prioritisation and provide comprehensive functional genomic evidence for the machineries, pathways and regulators that coordinate progression through the trypanosome cell cycle. The data can be searched and browsed using an interactive, open access, online data visualization tool (https://tryp-cycle.onrender.com).

genomics↗